Molecular characterization of a novel thermostable mannose-6-phosphate isomerase from Thermus thermophilus

Mannose-6-phosphate isomerase catalyzes the interconversion of mannose-6-phosphate and fructose-6-phosphate. The gene encoding a putative mannose-6-phosphate isomerase from Thermus thermophilus was cloned and expressed in Escherichia coli. The native enzyme was a 29 kDa monomer with activity maxima for mannose 6-phosphate at pH 7.0 and 80 °C in the presence of 0.5 mM Zn²⁺ that was present at one molecule per monomer. The half-lives of the enzyme at 65, 70, 75, 80, and 85 °C were 13, 6.5, 3.7, 1.8, and 0.2 h, respectively. The 15 putative active-site residues within 4.5 Å of the substrate mannose 6-phosphate in the homology model were individually replaced with other amino acids. The sequence alignments, activities, and kinetic analyses of the wild-type and mutant enzymes with amino acid changes at His50, Glu67, His122, and Glu132 as well as homology modeling suggested that these four residues are metal-binding residues and may be indirectly involved in catalysis. In the model, Arg11, Lys37, Gln48, Lys65 and Arg142 were located within 3 Å of the bound mannose 6-phosphate. Alanine substitutions of Gln48 as well as Arg142 resulted in increase of K_m and dramatic decrease of k_cat, and alanine substitutions of Arg11, Lys37, and Lys65 affected enzyme activity. These results suggest that these 5 residues are substrate-binding residues. Although Trp13 was located more than 3 Å from the substrate and may not interact directly with substrate or metal, the ring of Trp13 was essential for enzyme activity.     

Production of L-ribose from L-ribulose by a triple-site variant of mannose-6-phosphate isomerase from Geobacillus thermodenitrificans

A triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase from Geobacillus thermodenitrificans was obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (k(cat)/K(m)) for L-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co(2+). The triple-site variant produced 213 g/liter l-ribose from 300 g/liter L-ribulose for 60 min, with a volumetric productivity of 213 g liter(-1) h(-1), which was 4.5-fold higher than that of the wild-type enzyme. The k(cat)/K(m) and productivity of the triple-site variant were approximately 2-fold higher than those of the Thermus thermophilus R142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.     

Characterization of a mannose-6-phosphate isomerase from Geobacillus thermodenitrificans that converts monosaccharides

A recombinant mannose-6-phosphate isomerase from Geobacillus thermodenitrificans (GTMpi) isomerizes aldose substrates possessing hydroxyl groups oriented in the same direction at the C2 and C3 positions such as the d- and l-forms of ribose, lyxose, talose, mannose, and allose. The activity of GTMpi for d-lyxose isomerization was optimal at pH 7.0, 70°C and 1 mM Co²⁺. Under these conditions, the k _cat and K _m values were 74,300 s⁻¹ and 390 mM for d-lyxose and 28,800 s⁻¹ and 470 mM for l-ribose, respectively. The half-lives of the enzyme at 60, 65, and 70°C were 388, 73, and 27 h, respectively. GTMpi catalyzed the conversion of d-lyxose to d-xylulose with a 38% conversion yield after 3 h, and converted l-ribose to l-ribulose with a 29% conversion yield.     

Crystal structure of glucose-6-phosphate isomerase from Thermus thermophilus HB8 showing a snapshot of active dimeric state

Glucose-6-phosphate isomerase (GPI) is a glycolytic enzyme with ill-defined oligomeric state. In order to obtain insight into the correlation between oligomerization and the catalytic function of this enzyme, the crystal structure of GPI from the extreme thermophile Thermus thermophilus HB8 (TtGPI) has been determined at 1.95 Å resolution. The crystallographic asymmetric unit contains an apparent dimer. The core fold of protomer and the interprotomer spatial arrangement of the dimer are similar to those of already reported crystal structures of other GPIs. The active site is located on the dimer interface, and putative catalytic residues are well conserved among the GPIs. These results suggest that the observed dimeric state of TtGPI in the crystal is biologically relevant and that this enzyme uses a common catalytic mechanism for the isomerase reaction. Gel-filtration chromatography, chemical cross-linking, sedimentation equilibrium by analytical ultracentrifugation, and dynamic light-scattering experiments indicate that TtGPI exists in a dynamic equilibrium between monomeric and dimeric states in solution. Several factors potentially contributing to the thermal stability of TtGPI protomer were identified: (i) a decrease in denaturation entropy by the shorter polypeptide length and by amino acid composition, including the increased number of proline residues and a higher arginine-to-lysine ratio; (ii) a larger number of ion pairs; and (iii) a reduction in cavity volume. From these results, it is suggested that transient dimer formation is sufficient for the catalytic function and that the TtGPI protomer itself has intrinsically higher thermal stability.     

Characterization of recombinant L-ribose isomerase acquired from Cryobacterium sp. N21 with potential application in L-ribulose production

l-Ribose isomerase (lRI) is an enzyme that can catalyze the reversible isomerization between l-ribose and l-ribulose. It can also perform the conversion between many aldoses into their corresponding ketoses. l-RI was produced from Cryobacterium sp. N21 (CrL-RIse), and l-ribose was utilized as a substrate. The recombinant l-RI gene was cloned and overexpressed from Cryobacterium sp. N21. The purification of CrL-RIse was performed by metal-affinity chromatography. The enzyme displayed a corresponding band with an approximate size of 35 kDa on the SDS-PAGE analysis. The protein for this gene contains 266 amino acids with an expected molecular weight (M_w) of 29.6 kDa. The measured M_w of CrL-RIse calculated by HPLC was 125 kDa. CrL-RIse was extremely active in glycine buffer at 35 °C, pH 9.0, showing a specific activity of 54.96 U mg⁻¹. CrL-RIse displayed no major increase in activity with metal ions, excluding Mn²⁺. The estimated Km, K_cat, K_cat/Km and V_max values of CrL-RIse were 37.8 mM, 10,416 min⁻¹, 275.43 min⁻¹ mM⁻¹, and 250 U mg⁻¹, respectively. The rate of l-ribulose production was 31 % (6.24, 12.11, and 20.89 g L⁻¹) at equilibrium by utilizing 20, 40, and 70 g L⁻¹ of the substrate, respectively. The results indicated that CrL-RIse has the capability to manufacture l-ribulose from l-ribose.     

Identification of free mannose and partial characterization of a mannose-6-phosphate isomerase from Lilium longiflorum bulbs

The existence of free mannose in storage bulbs of Lilium longiflorum Thunb, was established using preparative high performance liquid chromatography, gas chromatography and gas chromatography-mass spectroscopy. Free mannose was not detected in developing (importing) bulb tissues. Mannose, a relatively rare hexose in plant tissue, probably arises from the hydrolysis of glucomannan, a hemicellulosic carbohydrate polymer known to be present in Lilium storage tissues. A calculation of total mannose residues per bulb (prior to versus after reserve hydrolysis and export) indicated that mannose is metabolized, probably in sucrose biosynthesis. A mannose-6-phosphate isomerase (EC 5.3.1.8) was isolated from Lilium bulbs and purified 155-fold with 29% yield. The molecular weight of the enzyme was estimated by gel filtration to be 64 kDa, and the K_m for mannose-6-phosphate was 0.42 m M . It is concluded that glucomannan is functioning as a reserve carbohydrate in Lilium storage tissues and that the mannose-6-phosphate isomerase is responsible for the entry of mannose into the sucrose biosynthetic pathway.     

Characterization of ornithine decarboxylase and regulation by its antizyme in Thermus thermophilus

Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis was highly purified from the thermophilic bacterium Thermus thermophilus. The enzyme preparation showed a single band on SDS-polyacrylamide gel electrophoresis, a pH optimum of 7.5 and a temperature optimum at 60°C. The native enzyme which is phosphorylated could, upon treatment with alkaline phosphatase, lose all activity. The inactive form could be reversibly activated by nucleotides in the order of NTP>NDP>NMP. When physiological polyamines were added to the purified enzyme in vitro, spermine or spermidine activated ODC by 140 or 40%, respectively, while putrescine caused a small inhibition. The basic amino acids lysine and arginine were competitive inhibitors of ODC, while histidine did not affect the enzyme activity. Among the phosphoamino acids tested, phosphoserine was the most effective activator of purified ODC. Polyamines added at high concentration to the medium resulted in a delay or in a complete inhibition of the growth of T. thermophilus, and in a decrease of the specific activity of ornithine decarboxylase. The decrease of ODC activity resulted from the appearance of a non-competitive inhibitor of ODC, the antizyme (Az). The T. thermophilus antizyme was purified by an ODC-Sepharose affinity column chromatography, as well as by immunoprecipitation using antibodies raised against the E. coli antizyme. The antizyme of E. coli inhibited the ODC of T. thermophilus, and vice versa. The fragment of amino acids 56-292 of the E. coli antizyme, produced as a fusion protein of glutathione S-transferase, did not inhibit the ODC of E. coli or T. thermophilus.     

Efficient production of thermostable Thermus thermophilus xylose isomerase in Escherichia coli and Bacillus brevis

Summary The xylose (glucose) isomerase from the thermophile Thermus thermophilus seems to have potential for the development of new isomerization processes using high temperatures and slightly acidic pH. The isomerase has an optimum temperature at 95° C, and is also very stable at high temperatures. The optimum pH is around 7.0, close to where by-product formation is minimal. Since Thermus produces only a little of this useful isomerase, the production of the cloned gene in Escherichia coli and Bacillus brevis were compared. Especially B. brevis was able to produce the isomerase effeciently, more than 1 g/l, in spite of the high G + content (67%) of the Thermus gene, and the presence of codons not frequently used in E. coli or B. brevis.Offprint requests to: S. Udaka     

Inhibition of hexose transport by glucose in a glucose-6-phosphate isomerase mutant ofSaccharomyces cerevisiae

The rate of hexose transport was approximately 60% lower for both the high- and the low-affinity components of hexose uptake when a glucose-6-phosphate isomerase mutant ofSaccharomyces cerevisiae was preincubated with glucose, as compared with preincubation with water. Similarly theJ _max value of the high-affinity system of the mutant was 25–35 % of the correspondingJ _max value for normal cells incubated with glucose. Accumulation of glucose 6-phosphate or of some other metabolite, such as fructose 6-phosphate or trehalose, may be responsible for this striking inhibition.     

Trehalose biosynthesis in Thermus thermophilus RQ-1: biochemical properties of the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase

The genes for trehalose synthesis in Thermus thermophilus RQ-1, namely otsA [trehalose-phosphate synthase (TPS)], otsB [trehalose-phosphate phosphatase (TPP)], and treS [trehalose synthase (maltose converting) (TreS)] genes are structurally linked. The TPS/TPP pathway plays a role in osmoadaptation, since mutants unable to synthesize trehalose via this pathway were less osmotolerant, in trehalose-deprived medium, than the wild-type strain. The otsA and otsB genes have now been individually cloned and overexpressed in Escherichia coli and the corresponding recombinant enzymes purified. The apparent molecular masses of TPS and TPP were 52 and 26 kDa, respectively. The recombinant TPS utilized UDP-glucose, TDP-glucose, ADP-glucose, or GDP-glucose, in this order as glucosyl donors, and glucose-6-phosphate as the glucosyl acceptor to produce trehalose-6-phosphate (T6P). The recombinant TPP catalyzed the dephosphorylation of T6P to trehalose. This enzyme also dephosphorylated G6P, and this activity was enhanced by NDP-glucose. TPS had an optimal activity at about 98°C and pH near 6.0; TPP had a maximal activity near 70°C and at pH 7.0. The enzymes were extremely thermostable: at 100°C, TPS had a half-life of 31 min, and TPP had a half-life of 40 min. The enzymes did not require the presence of divalent cations for activity; however, the presence of Co²⁺ and Mg²⁺ stimulates both TPS and TPP. This is the first report of the characterization of TPS and TPP from a thermophilic organism.     

Structure of Thermus thermophilus type 2 isopentenyl diphosphate isomerase inferred from crystallography and molecular dynamics

Crystal structures of Thermus thermophilus and Bacillus subtilis type 2 IPP isomerases were combined to generate an almost complete model of the FMN-bound structure of the enzyme. In contrast to previous studies, positions of flexible loops were obtained and carefully analyzed by molecular dynamics. Docking simulations find a unique putative binding site for the IPP substrate.     

Tetramer-dimer conversion of phosphofructokinase from Thermus thermophilus induced by its allosteric effectors

Phosphofructokinase (PFKase) was purified from an extreme thermophile. Thermus thermophilus. Allosteric natures of T. thermophilus PFKase is similar to those of Bacillus stearothermophilus PFKase, that is, hyperbolic plots of the activity versus concentration of fructose 6-phosphate (F6P) were changed into a sigmoidal shape by the addition of phosphoenolpyruvate (PEP), while further addition of ADP caused it to revert to a hyperbolic shape. The native T. thermophilus PFKase has an Mr of 148,000 consisting of four 36,500 subunits. However, it exists as a two-subunit form of Mr 74,000 in the presence of PEP. The two-subunit form was catalytically inactive. The four-subunit enzyme was regenerated by addition of either F6P or Mg.ADP, or by removal of PEP from the solution. This reversible dissociation was observed within a wide range of pH (6.5 to 8.4) and temperature (4 degrees C to 65 degrees C). Thus, unlike PFKase from other sources, the allosteric kinetics of T. thermophilus PFKase can be explained well, at least qualitatively, by the dynamic equilibrium between the active four-subunit form and inactive two-subunit form that is modulated by PEP, F6P and Mg.ADP. Parallel suppression of the PEP-induced conversion in molecular form and kinetics by high concentrations of sulfate and phosphate supports the above explanation. Also, the observation that the degree of PEP inhibition was dependent on the protein concentration of the PFKase in the assay solution is consistent with the presence of this equilibrium.     

Receptor-mediated uptake of a mannose-6-phosphate bearing glycoprotein by isolated chicken osteoclasts

We have recently shown that degradation of bone collagen by osteoclasts occurs via proteolytic enzyme activity that depends on an acidic milieu. Since bone resorption occurs in an extracellular, acidic compartment located at the cell-matrix attachment site, the osteoclast must deliver the acid collagenolytic enzymes to the cell surface. These observations raise the possibility that the mannose-6-phosphate (M-6-P) receptor, known to sort acidic proteases in other cells, is involved in trafficking lysosomal enzymes to the plasmalemma of bone resorbing cells. To this end we studied receptor-mediated uptake, distribution and release, by isolated chicken osteoclasts, of 125I-hexosaminidase, a M-6-P bearing enzyme. We found that at 4 degrees C, the bone-resorbing polykaryons bind approximately 10,000 molecules of radioligand/cell with a Kd of 0.7 nM, which is endocytosed by osteoclasts at 37 degrees C by a calcium-independent process. Furthermore, 125I-hexosaminidase uptake is unaffected by mannosylated albumin, documenting specificity of the receptor-mediated event. Release of endocytosed enzyme from the cell is also much more rapid than its degradation, attesting to a pathway of uptake and secretion. By autoradiography, the M-6-P bearing ligand is concentrated at the site of osteoclast-bone attachment. Thus, osteoclasts also have the capacity to deliver M-6-P bearing degradative enzymes to their surface at the site of matrix degradation.     

噬热栖热菌质粒分离蛋白同系物ParBTth的表达与重组酶活性

在低拷贝质粒中,质粒分离基因位点parABS对质粒DNA的子细胞精确分离有重要意义。噬热栖热菌(Thermus thermophilus)为多倍体,其染色体上表达有一个parABS同系物,命名为parABSTth,包含parATth和parBTth两个基因,以及一个候选parSTth的DNA序列(5’-TGTTTCCCGTGAAACA-3’)。parABSTth基因位点的功能与机制尚未清楚。本研究首先在大肠杆菌Escherichia coli中克隆并表达了T. thermophilus parABSTth位点中的parBTth基因。利用Ni-IDA亲和层析方法纯化得到了重组蛋白ParBTth;经SDS-PAGE测定,该重组蛋白纯度达到了近99%,通过凝胶迁移实验(EMSA)验证了重组ParBTth蛋白的功能。结果显示,重组ParBTth能与候选parSTthDNA序列特异性相结合。因此,异源表达的重组ParBTth蛋白是具有生物学活性的。该结果也验证了候选parSTthDNA序列确实为T. thermophilus的parABSTth基因位点中特异性的parS序列。本研究为揭示多倍体细菌染色体的子细胞分离过程原理提供了一定的基础理论依据。     

Osmotic adaptation of Thermus thermophilus RQ-1: lesson from a mutant deficient in synthesis of trehalose

Strains of Thermus thermophilus accumulate primarily trehalose and smaller amounts of mannosylglycerate in response to salt stress in yeast extract-containing media (O. C. Nunes, C. M. Manaia, M. S. da Costa, and H. Santos, Appl. Environ. Microbiol. 61:2351-2357, 1995). A 2.4-kbp DNA fragment from T. thermophilus strain RQ-1 carrying otsA (encoding trehalose-phosphate synthase [TPS]), otsB (encoding trehalose-phosphate phosphatase [TPP]), and a short sequence of the 5' end of treS (trehalose synthase [TreS]) was cloned from a gene library. The sequences of the three genes (including treS) were amplified by PCR and sequenced, revealing that the genes were structurally linked. To understand the role of trehalose during salt stress in T. thermophilus RQ-1, we constructed a mutant, designated RQ-1M6, in which TPS (otsA) and TPP (otsB) genes were disrupted by gene replacement. Mutant RQ-1M6 accumulated trehalose and mannosylglycerate in a medium containing yeast extract and NaCl. However, growth in a defined medium (without yeast extract, known to contain trehalose) containing NaCl led to the accumulation of mannosylglycerate but not trehalose. The deletion of otsA and otsB reduced the ability to grow in defined salt-containing medium, with the maximum salinity being 5% NaCl for RQ-1 and 3% NaCl for RQ-1M6. The lower salt tolerance observed in the mutant was relieved by the addition of trehalose to the growth media. In contrast to trehalose, the addition of glycine betaine, mannosylglycerate, maltose, and glucose to the growth medium did not allow the mutant to grow at higher salinities. The results presented here provide crucial evidence for the importance of the TPS/TPP pathway for the synthesis and accumulation of trehalose and the decisive contribution of this disaccharide to osmotic adaptation in T. thermophilus RQ-1.     

Characterization of a recombinant l-rhamnose isomerase from Dictyoglomus turgidum and its application for l-rhamnulose production

A putative recombinant enzyme from Dictyoglomus turgidum was characterized and immobilized on Duolite A568 beads. The native enzyme was a 46 kDa tetramer. Its activity was highest for l-rhamnose, indicating that it is an l-rhamnose isomerase. The maximum activities of both the free and immobilized enzymes for l-rhamnose isomerization were at pH 8.0 and 75 °C in the presence of Mn²⁺. Under these conditions, the half-lives of the free and immobilized enzymes were 28 and 112 h, respectively. In a packed-bed bioreactor, the immobilized enzyme produced an average of 130 g l-rhamnulose l^?1 from 300 g l-rhamnose l^?1 after 240 h at pH 8.0, 70 °C, and 0.6 h^?1, with a productivity of 78 g l^?1 h^?1 and a conversion yield of 43 %. To the best of our knowledge, this is the first report describing the enzymatic production of l-rhamnulose.     

Characterization and X-ray structure of the NADH-dependent coenzyme A disulfide reductase from Thermus thermophilus

The crystal structure of the enzyme previously characterized as a type-2 NADH:menaquinone oxidoreductase (NDH-2) from Thermus thermophilus has been solved at a resolution of 2.9 Å and revealed that this protein is, in fact, a coenzyme A-disulfide reductase (CoADR). Coenzyme A (CoASH) replaces glutathione as the major low molecular weight thiol in Thermus thermophilus and is maintained in the reduced state by this enzyme (CoADR). Although the enzyme does exhibit NADH:menadione oxidoreductase activity expected for NDH-2 enzymes, the specific activity with CoAD as an electron acceptor is about 5-fold higher than with menadione. Furthermore, the crystal structure contains coenzyme A covalently linked Cys44, a catalytic intermediate (Cys44-S-S-CoA) reduced by NADH via the FAD cofactor. Soaking the crystals with menadione shows that menadione can bind to a site near the redox active FAD, consistent with the observed NADH:menadione oxidoreductase activity. CoADRs from other species were also examined and shown to have measurable NADH:menadione oxidoreductase activity. Although a common feature of this family of enzymes, no biological relevance is proposed. The CoADR from T. thermophilus is a soluble homodimeric enzyme. Expression of the recombinant TtCoADR at high levels in E. coli results in a small fraction that co-purifies with the membrane fraction, which was used previously to isolate the enzyme wrongly identified as a membrane-bound NDH-2. It is concluded that T. thermophilus does not contain an authentic NDH-2 component in its aerobic respiratory chain.